Modified Leucine Dehydrogenase

ABSTRACT

The present invention provides a means and method useful for measurement of a total branched-chain amino acid concentration. Specifically, the present invention provides a modified enzyme in which at least one amino acid residue is mutated so as to improve a property of a leucine dehydrogenase which is associated with the measurement of the total branched-chain amino acids, such as, for example, substrate specificities of leucine dehydrogenase for total branched-chain amino acids, activity of leucine dehydrogenase for any branched-chain amino acids, and thermal stability of leucine dehydrogenase; and a method of analyzing the total branched-chain amino acids, comprising measuring the total branched-chain amino acids contained in a test sample using the modified enzyme.

This application is a continuation of, and claims priority under 35U.S.C. §120 to, International Patent Application No. PCT/JP2013/059124,filed on Mar. 27, 2013, which claims priority therethrough under 35U.S.C. §119 to Japanese Patent Application No. 2012-082777, filed onMar. 30, 2012, which are incorporated in their entireties by reference.Also, the Sequence Listing filed electronically herewith is herebyincorporated by reference (File name: 2014-09-19T_US-522_Seq_List; Filesize: 20 KB; Date recorded: Sep. 19, 2014).

FIELD OF THE INVENTION

The present invention related to a modified leucine dehydrogenase, amethod of analyzing total branched-chain amino acids using the same, andthe like.

BRIEF DESCRIPTION OF THE RELATED ART

It is known that some amino acids can become indicators for varioushealth conditions. In particular, branched-chain amino acids (BCAA:L-leucine, L-isoleucine and L-valine) are important amino acids that areabundant in various biological samples, foods, and beverages. Thebranched-chain amino acids are abundant in muscle in living bodies andare known as a marker of protein nutrition. It is known thatconcentrations of the branched-chain amino acids in blood are reduced inpatients with hepatic cirrhosis or hepatic encephalopathy, andindicators such as Fisher ratio and BTR value are utilized for healthcondition and follow-up of the liver.

Methods using analytical instruments, including high-performance liquidchromatography (HPLC) and LC-MS, are widely used to analyze amino acids.When measuring total branched-chain amino acids, an enzymatic kit formeasuring the BTR value utilizing leucine dehydrogenase (e.g., Japanesepatent application laid-open publication no. JP 2007-289096-A) and abiosensor for electrochemically measuring the total branched-chain aminoacids utilizing leucine dehydrogenase (e.g., International Publicationno. WO2005/075970) have been reported.

SUMMARY OF THE INVENTION

There are several points that could be improved in the measurement ofconcentrations of the total branched-chain amino acids using a leucinedehydrogenase.

For example, concentration of the total branched-chain amino acids ismeasured using a leucine dehydrogenase in an enzymatic kit. However,these methods typically are performed until an endpoint is reached atwhich the substrates are fully and completely reacted. This is becausesubstrate specificities (reaction rates) of the leucine dehydrogenasefor L-leucine, L-isoleucine and L-valine are each different. Forwild-type leucine dehydrogenase, the substrate specificities forL-isoleucine and L-valine are lower than that for L-leucine andtherefore the reaction rates for L-isoleucine and L-valine are slowerthan that for L-leucine. Therefore, existing methods have thedisadvantage that it takes a long time to measure the concentration ofthe total branched-chain amino acids in the enzymatic kit using theleucine dehydrogenase due to the longer reaction rates of L-isoleucineand L-valine.

In addition, when a plurality of amino acids that are the substrates ofthe leucine dehydrogenase are present, each concentration ofbranched-chain amino acids cannot be independently measured with thebiosensor. This is because the leucine dehydrogenase reacts not onlywith L-leucine but also with L-isoleucine and L-valine. In the case asabove, a total concentration of the branched-chain amino acids couldalso not be measured in general. This is because the substratespecificities (reaction rates) of the leucine dehydrogenase forL-leucine, L-isoleucine and L-valine are different from each other.

As a result of an extensive study, the present inventors have conceivedthat a concentration of total branched-chain amino acids may be measuredutilizing a rating method (initial rate method) by enhancing an activityof a leucine dehydrogenase for each amino acid of branched-chain aminoacids, particularly for L-isoleucine and L-valine, or the like, toimprove substrate specificities of the leucine dehydrogenase for thebranched-chain amino acids, in order to rapidly measure theconcentration of the total branched-chain amino acids, and havesucceeded in developing a modified leucine dehydrogenase that haimproved substrate specificities for the branched-chain amino acids. Thepresent inventors have also succeeded in improving other properties ofthe leucine dehydrogenase that are associated with the measurement ofthe concentration of the total branched-chain amino acids.

It is an aspect of the present invention to provide a modified leucinedehydrogenase enzyme comprising at least one amino acid mutation ascompared to a non-modified leucine dehydrogenase enzyme, wherein saidmodified leucine dehydrogenase is improved in one or more propertiesselected from the group consisting of:

(a) substrate specificities for total branched-chain amino acids;

(b) activity for any branched-chain amino acid;

(c) thermal stability; and

(d) combinations thereof.

It is a further aspect of the present invention to provide the modifiedleucine dehydrogenase enzyme as described above, wherein the mutation isa substitution of isoleucine in a TGI motif in an amino acid sequence ofthe non-modified leucine dehydrogenase enzyme.

It is a further aspect of the present invention to provide the modifiedleucine dehydrogenase enzyme as described above, wherein the isoleucinein the TGI motif is substituted with an amino acid selected from thegroup consisting of methionine, arginine, histidine, phenylalanine,leucine, lysine, cysteine, tyrosine, alanine, glycine, serine,asparagine, and tryptophan.

It is a further aspect of the present invention to provide the modifiedleucine dehydrogenase enzyme as described above, wherein the mutation isa substitution of isoleucine in a GVI motif in an amino acid sequence ofthe non-modified leucine dehydrogenase enzyme.

It is a further aspect of the present invention to provide the modifiedleucine dehydrogenase enzyme as described above, wherein the isoleucinein the GVI motif is substituted with an amino acid selected from thegroup consisting of phenylalanine, histidine, asparagine, tyrosine,leucine, lysine, glutamine, arginine, aspartic acid, threonine, glutamicacid, serine, cysteine, alanine, glycine, valine, tryptophan, andmethionine.

It is a further aspect of the present invention to provide the modifiedleucine dehydrogenase enzyme as described above, wherein thenon-modified leucine dehydrogenase enzyme is derived from Geobacillusstearothermophilus.

It is a further aspect of the present invention to provide the modifiedleucine dehydrogenase enzyme as described above, comprising a proteinselected from the group consisting of:

(A) a protein comprising the amino acid sequence of SEQ ID NO: 2, buthaving a substitution of isoleucine in the TGI motif with an amino acidresidue selected from the group consisting of methionine, arginine,histidine, phenylalanine, leucine, lysine, cysteine, tyrosine, alanine,glycine, serine, asparagine, or tryptophan;

(B) a protein comprising the amino acid sequence of SEQ ID NO: 2, buthaving a substitution of isoleucine in the GVI motif with an amino acidselected from the group consisting of phenylalanine, histidine,asparagine, tyrosine, leucine, lysine, glutamine, arginine, asparticacid, threonine, glutamic acid, serine, cysteine, alanine, glycine,valine, tryptophan, and methionine;

(C) a protein comprising the amino acid sequence of SEQ ID NO: 2, buthaving a substitution of isoleucine in the TGI motif with an amino acidselected from the group consisting of methionine, arginine, histidine,phenylalanine, leucine, lysine, cysteine, tyrosine, alanine, glycine,serine, asparagine, and tryptophan, and a substitution of isoleucine inthe GVI motif with an amino acid selected from the group consisting ofphenylalanine, histidine, asparagine, tyrosine, leucine, lysine,glutamine, arginine, aspartic acid, threonine, glutamic acid, serine,cysteine, alanine, glycine, valine, tryptophan, and methionine, and

(D) a protein as described in (A), (B), or (C) above, but alsocomprising one or several additional mutations of amino acid residues,and having one or more improved properties selected from the groupconsisting of:

(a) substrate specificities for total branched-chain amino acids;

(b) activity for any branched-chain amino acid; and

(c) thermal stability.

It is a further aspect of the present invention to provide a method ofanalyzing total branched-chain amino acids, comprising measuring thetotal branched-chain amino acids contained in a test sample using themodified leucine dehydrogenase enzyme as described above.

It is a further aspect of the present invention to provide the method asdescribed above, comprising mixing the test sample with nicotinamideadenine dinucleotide (NAD⁺) and detecting NADH formed from NAD⁺ by anaction of the modified leucine dehydrogenase enzyme.

It is a further aspect of the present invention to provide a method ofproducing a derivative of a branched-chain amino acid, comprisingforming the derivative from the branched-chain amino acid using themodified leucine dehydrogenase enzyme as described above.

It is a further aspect of the present invention to provide apolynucleotide encoding the modified leucine dehydrogenase enzyme asdescribed above.

It is a further aspect of the present invention to provide an expressionvector comprising the polynucleotide as described above.

It is a further aspect of the present invention to provide atransformant comprising the expression vector as described above.

It is a further aspect of the present invention to provide a method ofproducing a modified enzyme in which at least one amino acid residue ismutated so as to improve a property of a leucine dehydrogenase which isassociated with measurement of total branched-chain amino acids,comprising forming the modified enzyme using the transformant asdescribed above.

It is a further aspect of the present invention to provide a kit foranalyzing total branched-chain amino acids, comprising the modifiedenzyme as described above.

It is a further aspect of the present invention to provide the kit foras described above, further comprising at least one of a buffer solutionor a buffer salt for a reaction and nicotinamide adenine dinucleotide(NAD⁺).

It is a further aspect of the present invention to provide an enzymesensor for analyzing total branched-chain amino acids, comprising (a) anelectrode for detection and (b) the modified leucine dehydrogenaseenzyme as described above, which is immobilized or retained on theelectrode for detection.

The modified enzyme of the present invention is useful for rapidmeasurement of concentration of total branched-chain amino acids withimproved substrate specificity. The modified enzyme of the presentinvention is also useful for measurement of any branched-chain aminoacid and/or production of derivatives of any branched-chain amino acid(e.g., 2-oxo-derivative) because its activity for the branched-chainamino acids is enhanced. The modified enzyme of the present invention isalso excellent in stability because it is excellent in thermal stabilityin an aqueous solution. Therefore the modified enzyme of the presentinvention is useful particularly as a liquid reagent. The analysismethod of the present invention is useful for diagnosis of diseases suchas hepatic cirrhosis, hepatic encephalopathy, and the like.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows substrate specificities of each wild-type leucinedehydrogenase for each branched-chain amino acid (L-leucine,L-isoleucine and L-valine);

FIG. 2 shows changes of absorbance with time (means of n=3) when eachbranched-chain amino acid (L-leucine, L-isoleucine and L-valine) wasreacted with a wild-type enzyme or the modified enzyme I136R;

FIG. 3 shows activities of the wild-type enzyme or the modified enzymeI136R for each branched-chain amino acid (L-leucine, L-isoleucine andL-valine) at various concentration as changes of absorbance (means ofn=3) and showing standard curves prepared from those values; and

FIG. 4 shows concentrations of total BCAA (means of n=3) in plasmasamples from rats measured by an enzymatic method and an amino acidanalyzer.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention provides a modified enzyme. The modified enzyme ofthe present invention can be one in which at least one amino acidresidue in a leucine dehydrogenase is mutated so as to improve aproperty of the leucine dehydrogenase, which is associated with themeasurement of total branched-chain amino acids.

Examples of the mutation of the amino acid residue may includesubstitution, deletion, addition and insertion, and the substitution ispreferred particular example.

Amino acid residues to be mutated include L-alanine (A), L-asparagine(N), L-cysteine (C), L-glutamine (Q), glycine (G), L-isoleucine (I),L-leucine (L), L-methionine (M), L-phenylalanine (F), L-proline (P),L-serine (S), L-threonine (T), L-tryptophan (W), L-tyrosine (Y),L-valine (V), L-aspartic acid (D), L-glutamic acid (E), L-arginine (R),L-histidine (H) or L-lysine (K), and may be a naturally occurringL-α-amino acid. When the mutation is substitution, addition orinsertion, the amino acid residue to be substituted, added or insertedcan be the same as the amino acid residue to be mutated as describedabove.

The branched-chain amino acids (“BCAA”) include naturally occurringL-α-amino acids having a branched chain as a side chain, andspecifically include L-leucine, L-isoleucine, and L-valine. Thebranched-chain amino acids (i.e., L-leucine, L-isoleucine and L-valine)are collectively measured in the measurement of the total branched-chainamino acids. The leucine dehydrogenase is an oxidoreductase thatcatalyzes the following reaction (EC 1.4.1.9).

L-leucine+H₂O+NAD⁺→4-methyl-2-oxopentanoic acid+NH₃+NADH+H⁺

It is known that although a wild-type leucine dehydrogenase acts uponnot only L-leucine, but also on L-isoleucine and L-valine; although itsactivities for L-isoleucine and L-valine are lower than that forL-leucine. The substrate specificities of the wild-type leucinedehydrogenases derived from Bacillus sp. and Geobacillusstearothermophilus for L-leucine, L-isoleucine and L-valine are shown inFIG. 1 as relative activities when the relative activities for L-leucineare regarded as 100. As shown in FIG. 1, the activities of the wild-typeleucine dehydrogenases are relatively low for the branched-chain aminoacids other than L-leucine, and in particular, the relative activitiesfor L-isoleucine are 75 or less relative to the activities forL-leucine.

Wild-type leucine dehydrogenase derived from any organism, such asmicroorganisms such as bacteria, actinomycetes and fungi, as well asinsects, fish, animals and plants, can be used to derive the modifiedenzyme of the present invention. Examples of the wild-type leucinedehydrogenase may include those derived from organisms belonging to thegenus Bacillus and genera related thereto. Examples of the generarelated to the genus Bacillus may include the genus Geobacillus, thegenus Paenibacillus and the genus Oceanobacillus. The genera related tothe genus Bacillus belong to Bacillaceae, as is similar to the genusBacillus.

Examples of the microorganisms belonging to the genus Bacillus and thegenera related thereto may include Bacillus sphaericus, Bacillus cereus,Bacillus licheniformis, Bacillus sp., and Geobacillusstearothermophilus.

The position at which a mutation is introduced in the wild-type leucinedehydrogenase can be a position located in close proximity to an activecenter of the leucine dehydrogenase. A person skilled in the art canalign an amino acid sequence of the leucine dehydrogenase derived fromGeobacillus stearothermophilus with an amino acid sequence of anotherleucine dehydrogenase, and thus can easily determine an amino acidresidue position located in close proximity to an active center of thewild-type leucine dehydrogenases derived from organisms other thanGeobacillus stearothermophilus.

In addition, results of analyzing three-dimensional structures have beenreported for leucine dehydrogenases (see, e.g., Baker et al., Structure3: 693-705 (1995)). Therefore, a person skilled in the art can alsoeasily specify the amino acid residue located in close proximity of anactive center of the leucine dehydrogenases derived from organisms otherthan Geobacillus stearothermophilus, based on the results of analyzingthree-dimensional structure.

In a particular embodiment, a mutation that results in the improvementof the property of the leucine dehydrogenase associated with themeasurement of the total branched-chain amino acids can be asubstitution of isoleucine (I) in a TGI motif in the wild-type leucinedehydrogenase. The TGI motif is composed of the three consecutive aminoacid residues threonine (T)-glycine (G)-isoleucine (I). The position ofthe TGI motif in the amino acid sequence of the wild-type leucinedehydrogenase may be different depending on the origin of the enzyme.However, a person skilled in the art can appropriately determine theposition of the TGI motif in the amino acid sequence of the wild-typeleucine dehydrogenase, and thus can specify the position of isoleucine(I) to be substituted. Generally, in the amino acid sequence of thewild-type leucine dehydrogenase, the TGI motif is located within anamino acid region of positions 134 to 138 and the isoleucine (I) islocated at positions 136 to 138 (see, e.g., Table 1).

TABLE 1 Position of TGI motif in leucine dehydrogenase SEQ ID NO(sequence of wild-type enzyme Position Nucleotide Amino acid Leucinedehydrogenase TGI motif Ile sequence sequence G. stearothermophilus134-136 136 1 2 B. sphaericus 134-136 136 3 4 B. cereus 136-138 138 5 6B. licheniformis 134-136 136 7 8

In another particular embodiment, a mutation that results in theimprovement of the property of the leucine dehydrogenase associated withthe measurement of the total branched-chain amino acids is asubstitution of isoleucine (I) in a GVI motif of the wild-type leucinedehydrogenase. The GVI motif is composed of the three consecutive aminoacid residues of glycine (G)-valine (V)-isoleucine (I). The position ofthe GVI motif in the amino acid sequence of the wild-type leucinedehydrogenase may be different depending on the origin of the enzyme.However, a person skilled in the art can appropriately determine theposition of the GVI motif in the amino acid sequence of the wild-typeleucine dehydrogenase, and thus can specify the position of isoleucine(I) to be substituted. Generally, in the amino acid sequence of thewild-type leucine dehydrogenase, the GVI motif is located within anamino acid region at positions 290 to 294, and isoleucine (I) is locatedat positions 292 to 294 (see, e.g., Table 2). The modified enzyme of thepresent invention may further have the above substitution of isoleucine(I) in the TGI motif in addition to the substitution of isoleucine (I)in the GVI motif as the mutations to improve the property of the leucinedehydrogenase associated with the measurement of the totalbranched-chain amino acids.

TABLE 2 Position of GVI motif in leucine dehydrogenase SEQ ID NO(sequence of wild- type enzyme) Position Nucleotide Amino acid Leucinedehydrogenase GVI motif Ile sequence sequence G. stearothermophilus290-292 292 1 2 B. sphaericus 290-292 292 3 4 B. cereus 292-294 294 5 6B. licheniformis 290-292 292 7 8

The properties of the leucine dehydrogenase which are associated withthe measurement of the total branched-chain amino acids may include thefollowing:

(a) substrate specificities of the leucine dehydrogenase for the totalbranched-chain amino acids;

(b) an activity of the leucine dehydrogenase for any branched-chainamino acid; and

(c) a thermal stability of the leucine dehydrogenase.

The modified enzyme of the present invention may have only one of theaforementioned properties, or may have two or three of theaforementioned properties in combination.

For the isoleucine (I) in the TGI motif, examples of the mutation toimprove at least one property selected from the properties (a) to (c)may include substitutions with methionine (M), arginine (R), histidine(H), phenylalanine (F), leucine (L), lysine (K), cysteine (C), tyrosine(Y), alanine (A), glycine (G), serine (S), asparagine (N) and tryptophan(W).

For the isoleucine (I) in the GVI motif, examples of the mutation toimprove at least one property selected from the properties (a) to (c)may include substitutions with phenylalanine (F), histidine (H),asparagine (N), tyrosine (Y), leucine (L), lysine (K), glutamine (Q),arginine (R), aspartic acid (D), threonine (T), glutamic acid (E),serine (S), cysteine (C), alanine (A), glycine (G), valine (V),tryptophan (W) and methionine (M).

The modified enzyme of the present invention can be used under any pHcondition, and is suitably used under a neutral condition and/or analkaline condition.

The neutral condition under which the modified enzyme of the presentinvention is suitably used can be any pH condition within the range ofpH 6.0 or higher and pH 8.0 or lower. For example, the neutral conditioncan be any pH condition within the range of pH 7.0 or higher and pH 8.0or lower (e.g., pH 7.0, pH 7.5 or pH 8.0).

The alkaline condition under which the modified enzyme of the presentinvention is suitably used refers to any pH condition within the rangeof more than pH 8.0 and pH 11.0 or lower. An upper limit of a pH rangein the alkaline condition can be 10.5 or lower or even 10.0 or lower.For example, the alkaline condition can be any pH condition within therange of pH 8.5 or higher and pH 9.5 or lower (e.g., pH 9.0).

In one embodiment, the substrate specificities of leucine dehydrogenasefor the total branched-chain amino acids are improved as the property ofthe leucine dehydrogenase which is associated with the measurement ofthe total branched-chain amino acids. The improvement of the substratespecificities of the leucine dehydrogenase for the total branched-chainamino acids are not intended to enhance the substrate specificity of theleucine dehydrogenase for a certain branched-chain amino acid, butrefers to making the substrate specificities (reaction rates) for all ofthe branched-chain amino acids (i.e., L-leucine, L-isoleucine andL-valine) more equivalent. Specifically, the improvement of thesubstrate specificities of the leucine dehydrogenase for the totalbranched-chain amino acids can be accomplished when each relativeactivity of the modified enzyme for isoleucine and valine is closer to100 compared to each relative activity of the wild-type enzyme forisoleucine and valine, when the relative activity of the leucinedehydrogenase for leucine is regarded as 100. Concerning the substratespecificities of the leucine dehydrogenase for the total branched-chainamino acids, the relative activities of the leucine dehydrogenase forboth isoleucine and valine can be 80 or more and 120 or less, 85 or moreand 115 or less, 90 or more and 110 or less, or even 95 or more and 105or less when the relative activity of the leucine dehydrogenase forleucine is regarded as 100. Examples of the modification in the modifiedenzyme of the present invention in which the relative activities of theleucine dehydrogenase for both isoleucine and valine are 80 or more and120 or less when the relative activity for leucine is regarded as 100may include 1) the substitution of isoleucine (I) in the TGI motif withan amino acid residue described below and/or the substitution ofisoleucine (I) in the GVI motif with an amino acid residue describedbelow, which is suitable for the improvement of the substratespecificities under the alkaline condition, as well as 2) thesubstitution of isoleucine (I) in the TGI motif with an amino acidresidue described below and/or the substitution of isoleucine (I) in theGVI motif with an amino acid residue described below, which is suitablefor the improvement of the property under the neutral condition.

1) Substitution suitable for improvement of substrate specificitiesunder alkaline condition

(a) Amino acid residue after substitution at position 136 (alkalinecondition)

Methionine (M), arginine (R), phenylalanine (F), lysine (K), cysteine(C), tyrosine (Y), alanine (A), glycine (G) or serine (S)

(b) Amino acid residue after substitution at position 292 (alkalinecondition)

Phenylalanine (F), histidine (H), asparagine (N), tyrosine (Y), lysine(K), glutamine (Q), glutamic acid (E) or glycine (G)

2) Substitution suitable for improvement of substrate specificitiesunder neutral condition

(c) Amino acid residue after substitution at position 136 (neutralcondition)

Alanine (A), glycine (G), histidine (H), lysine (K), leucine (L), serine(S) or tyrosine (Y)

(d) Amino acid residue after substitution at position 292 (neutralcondition)

Alanine (A), cysteine (C), aspartic acid (D), glycine (G), lysine (K),leucine (L), methionine (M), arginine (R), serine (S), threonine (T) orvaline (V).

In another embodiment, the activity of the leucine dehydrogenase for anybranched-chain amino acid is improved as the property of the leucinedehydrogenase which is associated with the measurement of the totalbranched-chain amino acids. The improvement of the activity of theleucine dehydrogenase for any branched-chain amino acid means that theactivity of the modified enzyme for one or more amino acids such asL-leucine, L-isoleucine and L-valine is enhanced relative to theactivity of the wild-type enzyme for the same. Specifically, theimprovement of the activity of the leucine dehydrogenase for anybranched-chain amino acid can be accomplished in the case where theactivity of the modified leucine dehydrogenase for any amino acid suchas L-leucine, L-isoleucine and L-valine is higher than 100 when theactivity of the wild-type leucine dehydrogenase for that amino acid isregarded as 100. Such a modified enzyme enables rapid measurement of anindividual branched-chain amino acid, and consequently is useful for themeasurement of the total branched-chain amino acids. A level of theenhancement of the activity of the modified enzyme can be 1.3 fold ormore, 1.5 fold or more, 1.7 fold or more, or even 2.0 fold or morerelative to the activity of the wild-type enzyme. Examples of themodification in the modified enzyme of the present invention having 1.3fold or more enhancement of the activity relative to the wild-typeenzyme may include 1) the substitution of isoleucine (I) in the TGImotif with the following amino acid residue and/or the substitution ofisoleucine (I) in the GVI motif with the following amino acid residue,which is suitable for the improvement of the activity under the alkalinecondition as well as 2) the substitution of isoleucine (I) in the TGImotif with the following amino acid residue and/or the substitution ofisoleucine (I) in the GVI motif with the following amino acid residue,which is suitable for the improvement of the activity under the neutralcondition.

1) Substitution suitable for improvement of activity under alkalinecondition

1-1) Amino acid residues after substitution at position 136 (underalkaline condition)

(i) Enhancement of the activity for L-leucine (under alkaline condition)

Methionine (M), arginine (R), histidine (H), phenylalanine (F), leucine(L), lysine (K), cysteine (C), tyrosine (Y), alanine (A), glycine (G),serine (S), asparagine (N), or tryptophan (W).

(ii) Enhancement of activity for L-isoleucine (under alkaline condition)

Methionine (M), arginine (R), histidine (H), phenylalanine (F), leucine(L), lysine (K), cysteine (C), tyrosine (Y), alanine (A), glycine (G),serine (S), asparagine (N), or tryptophan (W).

(iii) Enhancement of activity for L-valine (under alkaline condition)

Methionine (M), arginine (R), histidine (H), phenylalanine (F), leucine(L), lysine (K), cysteine (C), tyrosine (Y), alanine (A), glycine (G),serine (S), asparagine (N), or tryptophan (W).

1-2) Amino acid residues after substitution at position 292 (underalkaline condition)

(iv) Enhancement of activity for L-leucine (under alkaline condition)

Phenylalanine (F), histidine (H), asparagine (N), tyrosine (Y), leucine(L), lysine (K), glutamine (Q), arginine (R), aspartic acid (D),threonine (T), glutamic acid (E), serine (S), cysteine (C), alanine (A),or glycine (G).

(v) Enhancement of activity for L-isoleucine (under alkaline condition)

Phenylalanine (F), histidine (H), asparagine (N), tyrosine (Y), leucine(L), lysine (K), glutamine (Q), arginine (R), aspartic acid (D),threonine (T), glutamic acid (E), serine (S), cysteine (C), alanine (A),glycine (G), valine (V), or tryptophan (W).

(vi) Enhancement of activity for L-valine (under alkaline condition)

Phenylalanine (F), histidine (H), asparagine (N), tyrosine (Y), leucine(L), lysine (K), glutamine (Q), arginine (R), aspartic acid (D),threonine (T), glutamic acid (E), serine (S), cysteine (C), alanine (A),glycine (G), or valine (V).

2) Substitutions suitable for improvement of activity under neutralcondition

2-1) Amino acid residues after substitution at position 136 (underneutral condition)

(i′) Enhancement of activity for L-leucine (under neutral condition)

Alanine (A), cysteine (C), phenylalanine (F), glycine (G), histidine(H), lysine (K), leucine (L), methionine (M), asparagine (N), arginine(R), serine (S), tryptophan (W), or tyrosine (Y).

(ii′) Enhancement of activity for L-isoleucine (under neutral condition)

Alanine (A), cysteine (C), phenylalanine (F), glycine (G), histidine(H), lysine (K), leucine (L), methionine (M), asparagine (N), arginine(R), serine (S), tryptophan (W), or tyrosine (Y).

(iii′) Enhancement of activity for L-valine (under neutral condition)

Alanine (A), cysteine (C), phenylalanine (F), glycine (G), histidine(H), lysine (K), leucine (L), methionine (M), asparagine (N), glutamine(Q), arginine (R), serine (S), tryptophan (W), or tyrosine (Y).

2-2) Amino acid residues after substitution at position 292 (underneutral condition)

(iv′) Enhancement of activity for L-leucine (under neutral condition)

Alanine (A), cysteine (C), aspartic acid (D), glutamic acid (E),phenylalanine (F), glycine (G), histidine (H), lysine (K), leucine (L),methionine (M), asparagine (N), glutamine (Q), arginine (R), serine (S),threonine (T), valine (V), tryptophan (W), or tyrosine (Y).

(v′) Enhancement of activity for L-isoleucine (under neutral condition)

Alanine (A), cysteine (C), aspartic acid (D), glutamic acid (E),phenylalanine (F), glycine (G), histidine (H), lysine (K), leucine (L),methionine (M), asparagine (N), glutamine (Q), arginine (R), serine (S),threonine (T), tryptophan (W), or tyrosine (Y).

(vi′) Enhancement of activity for L-valine (under neutral condition)

Alanine (A), cysteine (C), aspartic acid (D), glutamic acid (E),phenylalanine (F), glycine (G), histidine (H), lysine (K), leucine (L),methionine (M), asparagine (N), glutamine (Q), arginine (R), serine (S),threonine (T), valine (V), tryptophan (W), or tyrosine (Y).

In still another embodiment, the thermal stability of the leucinedehydrogenase is improved as the property of the leucine dehydrogenasewhich is associated with the measurement of the total branched-chainamino acids. The improvement of the thermal stability of the leucinedehydrogenase means that the thermal stability of the modified enzyme isfurther enhanced relative to that of the wild-type enzyme. Specifically,the improvement of the thermal stability of the leucine dehydrogenasecan be accomplished when a remaining activity of the modified enzyme ishigher than that of the wild-type enzyme when the enzyme is treated inan aqueous solution at 60° C. for one hour. A thermal stability test ofthe leucine dehydrogenase in the aqueous solution can have significanceas an acceleration test for evaluating the stability (particularlyliquid stability) of the leucine dehydrogenase. Therefore, when thethermal stability of the modified enzyme in the aqueous solution ishigh, the stability (particularly liquid stability) of the modifiedenzyme also tends to be high. An enzyme showing high liquid stabilitycan be stored in a liquid form for a long period of time, and thus, sucha modified enzyme is useful as a liquid reagent for the measurement ofthe total branched-chain amino acids. A level of the enhancement of thethermal stability of the modified enzyme can be 1.1 fold or more or 1.2fold or more relative to that of the wild-type enzyme. Examples of themodification in the modified enzyme of the present invention having 1.1fold or more enhanced thermal stability relative to the wild-type enzymemay include the substitution of isoleucine (I) in the TGI motif with thefollowing amino acid residue and/or the substitution of isoleucine (I)in the GVI motif with the following amino acid residue.

Amino acid residues after substitution at position 136

Methionine (M), arginine (R), phenylalanine (F), or lysine (K).

Amino acid residues after substitution at position 292

Phenylalanine (F)

The modified enzyme of the present invention may also have anotherpeptide component (e.g., a tag moiety) at the C-terminus or N-terminus.Examples of the other peptide component that can be added to themodified enzyme of the present invention may include peptide componentsthat make purification of the objective protein easy (e.g., tag moietysuch as histidine tag and strep-tag II; proteins such asglutathione-S-transferase and maltose-binding protein commonly used forthe purification of the objective protein), peptide components thatenhance solubility of the objective protein (e.g., Nus-tag), peptidecomponents that work as a chaperon (e.g., trigger factor), and peptidecomponents as a protein or a domain of the protein having anotherfunction or a linker connecting them.

The modified enzyme of the present invention may also have supplementalmutations (e.g., substitutions, deletions, insertions and additions) ofone or several amino acid residues in an amino acid sequence of theleucine dehydrogenase having the above mutation(s) as long as theaforementioned property is retained. The number of amino acid residuesin which the supplemental mutation can be introduced are, for example, 1to 100, 1 to 50, 1 to 40, 1 to 30 or even 1 to 20 or 1 to 10 (e.g., 1,2, 3, 4 or 5). A person skilled in the art can appropriately make such amodified enzyme retaining the aforementioned property.

Therefore, the modified enzyme of the present invention may be thefollowing (i) or (ii):

a protein having an amino acid sequence having a mutation or mutations(e.g., substitution) of isoleucine (I) in the TGI motif and/orisoleucine (I) in the GVI motif in an amino acid sequence of the leucinedehydrogenase, and having the improved property of the leucinedehydrogenase which is associated with the measurement of totalbranched-chain amino acids; or

a protein having an amino acid sequence having a supplemental mutationof one or several amino acid residues in the amino acid sequence havinga mutation or mutations (e.g., substitution) of isoleucine (I) in theTGI motif and/or isoleucine (I) in the GVI motif in the amino acidsequence of the leucine dehydrogenase, and having the improved propertyof the leucine dehydrogenase which is associated with the measurement ofthe total branched-chain amino acids.

The modified enzyme of the present invention may also be that having anamino acid sequence having at least 90% or more sequence identity to theamino acid sequence of the (wild-type) leucine dehydrogenase before itsmutation because of having both the aforementioned mutation or mutationsand the supplemental mutation or mutations. A percentage of the aminoacid sequence identity may be 92% or more, 95% or more, 97% or more, or98% or more or 99% or more.

The identity between the amino acid sequences can be determined, forexample, using algorithm BLAST by Karlin and Altschul (Pro. Natl. Acad.Sci. USA, 90, 5873 (1993)) and FASTA by Pearson (MethodsEnzymol., 183,63 (1990)). A program referred to as BLASTP has been developed based onthis algorithm BLAST (see http://www.ncbi.nlm.nih.gov). Thus, theidentity between the amino acid sequences may be calculated using theseprograms with the default setting. Also, for example, a numerical valueobtained by calculating similarity as a percentage using a full lengthpolypeptide portion encoded in an ORF and using software GENETYX Ver.7.09 with setting of Unit Size to Compare=2 from Genetyx Corporationemploying Lipman-Pearson method may be used as the identity between theamino acid sequences. The lowest value among the values derived fromthese calculations may be employed as the identity between the aminoacid sequences.

The position of an amino acid residue at which the supplemental mutationcan be introduced in an amino acid sequence would be apparent to aperson skilled in the art. For example, the supplemental mutation can beintroduced with reference to an alignment of the amino acid sequence.Specifically, a person skilled in the art can (1) compare amino acidsequences of a plurality of homologs (e.g., an amino acid sequencerepresented by SEQ ID NO:2 and an amino acid sequence of the otherhomolog(s)), (2) demonstrate relatively conserved regions and relativelynot conserved regions, then (3) predict regions capable of playing afunctionally important role and regions incapable of playing afunctionally important role from the relatively conserved regions andthe relatively not conserved regions, respectively, and thus recognizecorrelativity between a structure and a function. The analysis result ofthe three-dimensional structure has been reported for leucinedehydrogenases as described above. Thus, a person skilled in the art canintroduce the supplemental mutation based on the analysis result of thethree-dimensional structure so as to enable the retention of theaforementioned property.

When the supplemental mutation of the amino acid residue is asubstitution, such a substitution of the amino acid residue may be aconservative substitution. The term “conservative substitution” refersto substituting a given amino acid residue with an amino acid residuehaving a similar side chain. Families of the amino acid residues havingthe similar side chain are well-known in the art. Examples of suchfamilies may include amino acids having a basic side chain (e.g.,lysine, arginine, histidine), amino acids having an acidic side chain(e.g., aspartic acid, glutamic acid), amino acids having an unchargedpolar side chain (e.g., asparagine, glutamine, serine, threonine,tyrosine, cysteine), amino acids having a nonpolar side chain (e.g.,glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan), amino acids having a branched side chain atposition β (e.g., threonine, valine, isoleucine), amino acids having anaromatic side chain (e.g., tyrosine, phenylalanine, tryptophan,histidine), amino acids having a side chain containing a hydroxyl (e.g.,alcoholic, phenolic) group (e.g., serine, threonine, tyrosine), andamino acids having a sulfur-containing side chain (e.g., cysteine,methionine). For example, the conservative substitution of the aminoacid may be the substitution between aspartic acid and glutamic acid,the substitution between arginine, lysine and histidine, thesubstitution between tryptophan and phenylalanine, the substitutionbetween phenylalanine and valine, the substitution between leucine,isoleucine and alanine, and the substitution between glycine andalanine.

The modified enzyme of the present invention can be prepared using atransformant of the present invention, which expresses the modifiedenzyme of the present invention or a cell-free system. The transformantof the present invention can be made, for example, by making anexpression vector for the modified enzyme of the present invention andintroducing this expression vector into a host. For example, thetransformant of the present invention can be obtained by making theexpression vector in which the polynucleotide of the present inventionhas been incorporated and introducing this vector into an appropriatehost. Various prokaryotic cells including cells from bacteria belongingto genera Escherichia (e.g., Escherichia coli), Corynebacterium (e.g.,Corynebacterium glutamicum) and Bacillus (e.g., Bacillus subtilis), andvarious eukaryotic cells including cells from fungi belonging to generaSaccharomyces (e.g., Saccharomyces cerevisiae), Pichia (e.g., Pichiastipitis) and Aspergillus (e.g., Aspergillus oryzae) can be used as thehost for expressing the modified enzyme of the present invention. Astrain in which a certain gene has been deleted may be used as the host.Examples of the transformant may include transformants in which thevector is retained in its cytoplasm and transformants in which anobjective gene is integrated into its genome.

The transformant of the present invention can be cultured in a mediumhaving a composition described later using a given culture apparatus(e.g., test tube, flask, jar fermenter). A culture condition canappropriately be determined. Specifically, a culture temperature may be25 to 37° C., a pH value may be 6.5 to 7.5, and a culture period may be1 to 100 hours. Cultivation may also be carried out by managing thedissolved oxygen concentration. In this case, the dissolved oxygenconcentration (DO value) may be used as an indicator for control. Aventilation/stirring condition can be controlled so that the relativedissolved oxygen concentration, the DO value, does not fall below 1 to10% for example, or not below 3 to 8% when an oxygen concentration inthe air is 21%. The cultivation may be a batch cultivation or afed-batch cultivation. In the case of the fed-batch cultivation, thecultivation can also be continued by sequentially adding continuously ordiscontinuously a solution as a sugar source and a solution containingphosphoric acid to the culture medium.

The host to be transformed is as described above, and can be Escherichiacoli, or the host can be Escherichia coli K12 subspecies Escherichiacoli JM109 strain, DH5α strain, HB101 strain, BL21 (DE3) strain, and thelike. Methods of performing the transformation and methods of selectingthe transformant have been described in Molecular Cloning: A LaboratoryManual, 3rd edition, Cold Spring Harbor press (Jan. 15, 2001), and thelike. Hereinafter, a method of making transformed Escherichia coli andproducing a predetermined enzyme using this will be describedspecifically by way of example only.

A promoter used for producing a foreign protein in E. coli can generallybe used as a promoter for expressing the polynucleotide of the presentinvention. Examples thereof may include potent promoters such as a PhoA,PhoC, T7 promoter, a lac promoter, a trp promoter, a trc promoter, a tacpromoter, PR and PL promoters of lambda phage, and a T5 promoter, andthe PhoA, PhoC and lac promoters are preferred. For example, pUC (e.g.,pUC19, pUC18), pSTV, pBR (e.g., pBR322), pHSG (e.g., pHSG299, pHSG298,pHSG399, pHSG398), RSF (e.g., RSF1010), pACYC (e.g., pACYC177,pACYC184), pMW (e.g., pMW119, pMW118, pMW219, pMW218), pQE (e.g., pQE30)and derivatives thereof may be used as a vector. A vector from phage DNAmay also be utilized as the other vector. Further, an expression vectorthat includes a promoter and can express an inserted DNA sequence mayalso be used. Preferably, the vector may be pUC, pSTV, or pMW.

Also, a terminator that is a transcription terminating sequence may beligated downstream of the polynucleotide of the present invention.Examples of such a terminator may include a T7 terminator, an fd phageterminator, a T4 terminator, a terminator of a tetracycline resistantgene, and a terminator of Escherichia coli trpA gene.

The vector for introducing the polynucleotide of the present inventioninto Escherichia coli can be a so-called multicopy type, and examplesthereof may include plasmids which have an replication origin fromCo1E1, such as pUC-based plasmids, pBR322-based plasmids and derivativesthereof. Here the “derivative” means those in which the modification hasbeen given to the plasmid by substitution, deletion, insertion and/oraddition of base(s). The “modification” referred to herein also includesmodification by mutagenesis using a mutating agent, UV irradiation orthe like, or naturally occurring mutations.

In order to select the transformant, the vector can have a marker suchas an ampicillin resistant gene. Expression vectors having a potentpromoter are commercially available as such a plasmid (e.g., pUC-based(supplied from Takara Bio Inc.), pPROK-based (supplied from Clontech),pKK233-2-based (supplied from Clontech)).

The modified enzyme of the present invention can be obtained bytransforming Escherichia coli using the resulting expression vector ofthe present invention and culturing this Escherichia coli.

Media such as M9/casamino acid medium and LB medium generally used forculturing Escherichia coli may be used as the medium. The medium maycontain a predetermined carbon source, nitrogen source, and coenzyme(e.g., pyridoxine hydrochloride). Specifically, peptone, yeast extract,NaCl, glucose, MgSO₄, ammonium sulfate, potassium dihydrogen phosphate,ferric sulfate, manganese sulfate, and the like may be used. Acultivation condition and a production inducing condition areappropriately selected depending on types of a marker and a promoter ina vector and a host to be used.

The modified enzyme of the present invention can be recovered by thefollowing methods. The modified enzyme of the present invention can beobtained as a pulverized or lysed product by collecting the transformantof the present invention and subsequently pulverizing (e.g., sonicationor homogenization) or lysing (e.g., treatment with lysozyme) themicrobial cells. The modified enzyme of the present invention can beobtained by subjecting such a pulverized or lysed product to techniquessuch as extraction, precipitation, filtration, and columnchromatography.

The present invention also provides a method of analyzing the totalbranched-chain amino acids. The analysis method of the present inventioncan include the steps of measuring the total branched-chain amino acidscontained in a test sample using the modified enzyme of the presentinvention.

The test sample is not particularly limited as long as the sample issuspected of containing any branched-chain amino acid (preferably thetotal branched-chain amino acids), and examples thereof may includebiological samples (e.g., blood, urine, saliva, tear, and the like) andfood and beverage (e.g., nutrient drinks and amino acid beverages).

The analysis method of the present invention is not particularly limitedas long as the total branched-chain amino acids can be measured usingthe modified enzyme of the present invention. For example, the totalbranched-chain amino acids can be measured by mixing the test samplewith nicotinamide adenine dinucleotide (NAD⁺) under the alkalinecondition or the neutral condition, preferably in alkaline buffer, thensubjecting the mixed sample to an enzymatic reaction using the modifiedenzyme of the present invention, and finally detecting NADH formed fromNAD⁺ by the action of the modified enzyme of the present invention.Specifically, by allowing the modified enzyme to act upon the testsample in the alkaline buffer in the presence of nicotinamide adeninedinucleotide (NAD⁺), the reduced form (NADH) is generated fromnicotinamide adenine dinucleotide (NAD⁺) while an amino group of asubstrate contained in the biological sample is oxidatively deaminated.Thus, the total branched-chain amino acids can be quantified bydetecting NADH by an absorbance (340 nm) or the like. The methods ofmeasuring the amino acid by such a methodology are known (see e.g.,Ueatrongchit T, Asano Y, Anal Biochem., 2011 Mar. 1; 410(1): 44-56). Thetotal branched-chain amino acids can also be quantified by reducing adye with the formed NADH and detecting color development of the reduceddye as an absorbance or the like. Further, NADH can also be detected byan electrochemical technique. For example, it is possible to measure thetotal branched-chain amino acids by electrochemically oxidizing NADHformed by allowing the modified enzyme to act upon the test sample underthe alkaline or neutral condition and measuring its oxidation electriccurrent, or by reducing a coexisting electronic mediator by the formedNADH and measuring oxidation electric current when the reducedelectronic mediator is electrochemically oxidized. An electronictransfer between the NADH and the electronic mediator may be mediated bya catalyst. The total branched-chain amino acids can be measured by therating method (initial rate method).

The modified enzyme of the present invention is not reacted with aminoacids other than the branched-chain amino acids or has a low reactivitytherewith. Therefore, even when not only the branched-chain amino acidsbut also other amino acids are contained in a test sample, an amount ofthe branched-chain amino acids in the test sample can be evaluated byusing the modified enzyme of the present invention.

Further, the present invention includes a kit for analyzing the totalbranched-chain amino acids including the modified enzyme of the presentinvention.

The kit of the present invention can further include at least one of abuffer solution or a buffer salt for a reaction and nicotinamide adeninedinucleotide (NAD⁺).

The buffer solution or the buffer salt for the reaction is used forkeeping a pH value in a reaction solution suitable for an objectiveenzymatic reaction. The buffer solution or the buffer salt for thereaction is alkaline or neutral, and preferably alkaline.

When the kit of the present invention includes nicotinamide adeninedinucleotide (NAD⁺), the kit of the present invention may furtherinclude a dye to be reduced by NADH. In this case, the dye is reduced byNADH formed from NAD⁺ by an action of the modified enzyme of the presentinvention, and the color development from the reduced dye can bedetected by the absorbance and the like. A substance working as anelectronic mediator may be involved in the reduction of the dye.

The present invention also provides an enzyme sensor for analyzing thebranched-chain amino acid including (a) an electrode for detection and(b) the modified enzyme of the present invention immobilized or retainedon the electrode for detection. The modified enzyme of the presentinvention is immobilized or retained on the electrode directly orindirectly.

It is possible to use, for example, a biosensor that directly orindirectly detects a product or a byproduct (NH₃+NADH+H⁺) formed formthe total branched-chain amino acids by the modified enzyme of thepresent invention as the electrode for detection. More specifically,examples of the electrode for detection include an electrode fordetection utilizing the modified enzyme of the present invention andnicotinamide adenine dinucleotide (NAD⁺). Those described inInternational Publication No. WO2005/075970 and InternationalPublication No. WO00/57166 or others can be used as such an electrodefor detection.

EXAMPLES

The present invention will be described in detail with reference tofollowing Examples, but the present invention is not limited thereto.

Enzymatic Quantification Method

In an enzymatic quantification method for L-leucine, L-isoleucine andL-valine, leucine dehydrogenase was allowed to act upon a biologicalsample (e.g., plasma) in alkaline buffer in the presence of nicotinamideadenine dinucleotide (NAD⁺), and an amino group in a substrate containedin the biological sample was oxidatively deaminated, as well as areduced product (NADH) was formed from nicotinamide adenine dinucleotide(NAD⁺). The amount of NADH that formed was measured using a microplatereader (SpectraMax M2e, supplied from Molecular Devices).

Example 1 Production of Modified Enzyme (I136R)

(1) Preparation of Template 1dh Gene

(a) Culture and Purification of Chromosomal DNA

A lyophilized pellet of Geobacillus stearothermophilus NBRC 12550obtained from National Institute of Technology and Evaluation,Biological Resource Center (NBRC) was suspended in a growth medium 702,which was then applied onto an agar medium of a growth medium 802 andcultured overnight at 50° C. A resulting colony was inoculated in 5 mLof the growth medium 702 and statically cultured at 50° C. for 30 hours.Chromosomal DNA was purified from 5 mL of this cell culture, andsubjected to the following experiment.

Details for preparing the media are as described in Table 3.

TABLE 3 Composition of media Growth medium 702 Polypeptone 10 g  Yeastextract 2 g MgSO₄•7H₂O 1 g Growth medium 802 Polypeptone 10 g  Yeastextract 2 g MgSO₄•7H₂O 1 g Agar 15 g  Distilled water 1 L (pH 7.0)

(b) Preparation of Plasmid

pUC18His plasmid made by the method described in Biosci. Biotechnol.Biochem. 2009; 73: 729-732 was purified from a culture medium ofrecombinant Escherichia coli (E. coli JM109/pUC18His) carrying a vectorusing Invisorb Spin Plasmid Mini Kit (supplied from Invitek) accordingto manufacturer's protocol. Subsequently, 7 μL of vector DNA of purifiedplasmid pUC18His, 2.5 μL of 10×K buffer (supplied from Takara Bio Inc.)and each 0.8 μL of PstI and BamHI were mixed, sterilized ultrapure waterwas added to make a total volume of a reaction solution 25 μL, and thenthe mixture was treated with the restriction enzymes at 37° C. for 3hours. Then, 2 μL of alkaline phosphatase derived from shrimp (suppliedfrom Roche) and 5 μL of ×10 alkaline phosphatase buffer were added to 25μL of the above reaction solution, and sterilized ultrapure water wasadded to make a total volume of a reaction solution 50 μL, and then themixture was reacted at 37° C. for one hour. The reaction solution waspurified by phenol/chloroform extraction and ethanol precipitation toobtain 20 μL of dephosphorylated pUC18His vector DNA dissolved in a TEsolution.

(c) Amplification of Leucine Dehydrogenase Gene

G. stearothermophilus chromosomal DNA was used as a template, and asynthesized oligonucleotide primer 1:5′-CCGGATCCGATGGAATTGTTCAAATATATGGAAAC-3′ (SEQ ID NO:11) (supplied fromHokkaido System Science Co., Ltd) containing a BamHI recognition sitesequence and a synthesized oligonucleotide primer 2:5′-ACTGCAGTTATATTGCCGAAGCACC-3′ (SEQ ID NO:12) (supplied from HokkaidoSystem Science Co., Ltd) containing a PstI recognition site sequence,which had been both made based on a leucine dehydrogenase gene from G.stearothermophilus IFO 12550 (SEQ ID NO: 1; Biochemistry, 27, 9056(1988)) were used in amplification of the leucine dehydrogenase genederived from G. stearothermophilus. As a PCR reaction solution, 50 ng ofchromosomal DNA, each 1 μL of the synthesized nucleotide primers at 100pmol/μL, 5 μL of ExTaq ×10 buffer (supplied from Takara Bio Inc.), 5 μLof 2.5 mM dNTP mixture (supplied from Takara Bio Inc.), and 1 μL ofTaKaRa ExTaq DNA polymerase (supplied from Takara Bio Inc.) were mixed,and sterilized ultrapure water was added to make a total volume of thereaction solution 50 μL. PCR was performed using PTC-200 Peltier thermalcycler (supplied from MJ Research Japan), and the reaction at 94° C. for30 seconds, 55° C. for 30 seconds and 72° C. for 2 minutes was repeatedin 30 cycles.

A PCR product was electrophoresed, and two amplified products (around1400 by and 1900 bp) cut out under ultraviolet irradiation was extractedand purified using a gel extraction kit Gel-M™ Gel Extraction Kit(supplied from VIOGENE) to obtain 50 μL of a purified product.Subsequently, 6 μL of 10×K buffer (supplied from Takara Bio Inc.) andeach 1 μL of PstI and BamHI were added to 4 μL of the purified product,and sterilized ultrapure water was added to make a total volume of thereaction solution 60 μL. The reaction solution was reacted at 37° C. for3 hours to treat both ends of the purified PCR product with therestriction enzymes. The reaction solution was incubated at 60° C. for15 minutes to inactivate the restriction enzymes. Subsequently theethanol precipitation was performed to obtain a purified insertionfragment.

(d) Ligation and Transformation

1 μL of the dephosphorylated plasmid obtained above, 5 μL of thepurified insertion fragment, and 6 μL of Ligation Mix (supplied fromTakara Bio Inc.) were mixed, a total volume of a reaction solution wasmade 12 μL, and a ligation reaction was performed at 16° C. overnight.Subsequently, E. coli JM109 was transformed with 6 μL of the reactionsolution after the ligation reaction, applied onto an LB agar mediumcontaining 50 μg/mL of ampicillin, and cultured at 37° C. for 10 hours.A resulting colony was inoculated to a master plate and cultured, andthen a newly formed colony was inoculated to 5 mL of the LB agar mediumcontaining 50 μg/mL of ampicillin and cultured overnight. Plasmid DNAwas purified from the culture, and its nucleotide sequence was analyzedusing ABI PRISM 310 Genetic Analyzer (supplied from Applied Biosystems).A clone confirmed to have the correct nucleotide sequence of the leucinedehydrogenase gene derived from G. stearothermophilus was designated asE. coli JM109/pUCHisLDH.

(2) Production of Modified Enzyme of Leucine Dehydrogenase Derived fromGeobacillus stearothermophilus

An expression plasmid for a modified enzyme (I136R) was made byintroducing a site-directed mutation into pUCHisLDH using QuikChangeLightning Site-Directed Mutagenesis Kits (Agilent Technologies)according to the protocol attached to the product. At that time, asequence: 5′-GACTATGTCACCGGCCGTTCGCCCGAATTCGG-3′ (SEQ ID NO:9) and asequence: 5′-CCGAATTCGGGCGAACGGCCGGTGACATAGTC-3′ (SEQ ID NO:10) wereused as a sense primer containing a mutated codon and an antisenseprimer, respectively. Experimental manipulations associated withtransformation, cultivation, plasmid extraction, and the like werecarried out according to standard methods. A clone identified to have anobjective nucleotide sequence was designated as Escherichia coliJM109/pUCHisLDH mutant and used for subsequent experiments.

(3) Expression and Purification of Modified Enzyme

Expression and purification of the modified enzyme constructed asdescribed previously are shown below. A colony of recombinantEscherichia coli transformed with the plasmid pUCHisLDH mutantcontaining the gene encoding the modified enzyme (Escherichia coliJM109/pUCHisLDH mutant) was cultured with shaking in a test tubecontaining 5 mL of the LB medium containing 50 μg/mL of ampicillin saltat 37° C. for 16 hours. This culture was inoculated to a 0.5 literSakaguchi flask containing 100 mL of the LB medium containing 50 μg/mLof ampicillin salt, and cultured with shaking at 37° C. until O.D.reached 1.0. Then, 1 M isopropyl-β-D-galactoside (IPTG supplied fromNacalai Tesque Inc.) was added at a final concentration of 1 mM, and thecultivation with shaking was further continued for additional 5 hours.The resulting cultured medium was centrifuged (8,000 rpm, 15 minutes, 4°C.; Hitachi high speed cooled centrifuge, HIMAC CR21G supplied fromHitachi Ltd.) to precipitate cultured microbial cells, and a supernatantwas discarded. The obtained microbial cells were suspended in 50 mMNaH₂PO₄ buffer (pH 8.0) containing 300 mM NaCl and 10 mM imidazole.Then, the microbial cells were disrupted and the enzyme was extractedusing an ultrasonic disruption apparatus (KUBOTA INSONATOR model 201M,supplied from Kubota Corporation) at 180 W for 15 minutes at 4° C. Acell lysate was centrifuged (8,000 rpm, 15 minutes, 4° C.; Hitachi highspeed cooled centrifuge, HIMAC CR21G supplied from Hitachi Ltd.) and asupernatant was used as a cell-free extract solution for thepurification of an enzymatic protein. The cell-free extract solution wasapplied onto a column filled with 1 mL of Ni-NTA resin (supplied fromQiagen) and equilibrated with buffer (50 mM NaH₂PO₄ buffer (pH 8.0)containing 300 mM NaCl and 10 mM imidazole). The resin was washed with 5mL of washing buffer (50 mM NaH₂PO₄ buffer (pH 6.5) containing 1 M NaCland 20 mM imidazole), and subsequently a bound enzymatic protein waseluted with 5 mL of elution buffer (50 mM NaH₂PO₄ buffer (pH 8.0)containing 300 mM NaCl and 250 mM imidazole). The eluted fraction wasconcentrated using an ultrafiltration membrane (e.g., Vivaspin6 100 kDaMWCO supplied from GE Healthcare). A protein concentration was measuredusing Micro BCA Protein Assay Kit (supplied from Thermo FisherScientific) and calculated based on a standard curve prepared usingpredetermined concentration of bovine serum albumin. A purity of thepurified enzyme was confirmed by sodium dodecyl sulfate/polyacrylamidegel electrophoresis. Subsequent experiments were carried out using theobtained purified enzyme.

Example 2 Evaluation of Substrate Specificity

An activity of leucine dehydrogenase was measured in a cuvette with 1 cmof an optical pass length (supplied from Bio-Rad) using a microplatereader also capable of using a cuvette (SpectraMax M2e, supplied fromMolecular Devices) according to Asano et al.'s method (Eur. J. Biochem.(1987) 168 (1), 153-159). A composition of a reaction solution was asfollows. 0.5 mL of 0.2 M Glycine-KCl-KOH buffer (pH9.0), 0.04 mL of asolution of 25 mM NAD (supplied from Sigma), and 0.1 mL of a solution of0.1 M L-leucine (supplied from Sigma) or L-isoleucine (supplied fromSigma) or L-valine (supplied from Sigma) and an appropriate amount of anenzyme solution were added to make a total volume of the reactionsolution 1.0 mL. An enzymatic reaction was performed at room temperaturefor one minute, and change of an absorbance at 340 nm was measured. Theresults are shown in FIG. 2. A relative activity of the modified enzyme(I136R) to which the mutation had been introduced using the wild-type(WT) as the template was improved for each BCAA. Hereinafter, thewild-type is sometimes abbreviated as WT.

Example 3 Preparation of Standard Curve for Reaction of BCAA withModified Enzyme (I136R)

0.5 mL of 0.2 M Glycine-KCl-KOH buffer (pH9.0), 0.04 mL of a solution of25 mM NAD⁺ (supplied from Sigma), and a known concentration of anL-leucine or L-isoleucine or L-valine aqueous solution were mixed, andMilliQ water was added to make a total volume of 0.96 mL. This mixturewas added to a cuvette. To this solution, 0.04 mL of 0.5 mg/mL enzymesolution was added to make a total volume of 1 mL, and the resultingsolution was mixed upside down. The enzymatic reaction was performed atroom temperature for one minute, and the change of the absorbance at 340nm was measured.

The results of the above measurement are shown in FIG. 3. When BCAAconcentrations were 140 to 560 μM (measured at 4 points of 140, 280, 420and 560 μM), the standard curves for Leu, Ile and Val were largelydifferent in the case of WT because the activity of WT for each BCAA wasdifferent. On the other hand, the standard curves for the reaction ofLeu, Ile and Val with the modified enzyme I136R were almost the samebecause its activity for each BCAA was almost the same. From the above,it was revealed that according to the modified enzyme I136R, the totalBCAA concentration in even a sample containing any two or all of Leu,Ile and Val in mixture could be considered as concentration of any oneof Leu, Ile and Val.

Example 4 Quantification of Total BCAA in Actual Sample by ModifiedEnzyme I136R

Rat plasma samples (SD strain, females, 20 weeks of age, supplied fromCharles River Laboratories Japan, Inc.) were used as actual samples, andtotal BCAA in the sample was measured by LeuDH WT and LeuDH I136R. Themeasurement was evaluated by comparing total BCAA measurement valuescalculated by the enzymatic method with total BCAA measurement valuesobtained by an amino acid analyzer L-8900 (supplied from Hitachi HighTechnologies Corporation).

The quantification of total BCAA by the enzymatic method was carried outaccording to the following procedure. 0.5 mL of 0.2 M Glycine-KCl-KOHbuffer (pH9.0), 0.04 mL of a solution of 25 mM NAD⁺ (supplied fromSigma), and 0.04 mL or 0.02 mL of a known concentration of an L-valineaqueous solution or a rat plasma sample were mixed, and MilliQ water wasadded to make a total volume of 0.96 mL. This mixture was added to acuvette. To this solution, 0.04 mL of 0.5 mg/mL enzyme solution wasadded to make a total volume of 1 mL, and the resulting solution wasmixed upside down. The enzymatic reaction was performed at roomtemperature for one minute, and the change of the absorbance at 340 nmwas measured. The total BCAA in the plasma sample was quantified using astandard curve made by using the L-valine aqueous solution.

Deproteinization by sulfosalicylic acid was performed in the rat plasmasamples for measuring by the amino acid analyzer.

A graph comparing respective measurement values of the total BCAAconcentration in the rat plasma samples obtained by the enzymatic methodand by the amino acid analyzer is shown in FIG. 4. As a result, themeasurement values of the total BCAA concentration in the actual samplesobtained by the enzymatic method were almost the same as the measurementvalues of the total BCAA obtained by the amino acid analyzer. Thewild-type leucine dehydrogenase is known to scarcely have a catalyticactivity for amino acids other than the branched-chain amino acids. TheBCAA in the plasma sample containing a plurality of amino acids could bequantified in this experiment. Thus, the modified enzyme of the presentinvention is predicted to retain the property of the wild-type enzymethat the wild-type enzyme scarcely has the catalytic activity for theamino acids other than the branched chain amino acids. As describedabove, it was demonstrated that the modified enzyme of the presentinvention was useful for the measurement specific for the total BCAA inthe actual sample.

Example 5 Synthesis of Modified Enzyme Using Cell-Free System andPurification of Modified Enzyme

A histidine affinity tag and a TEV protease recognition site were fusedto an N terminal side by a 2-step PCR method using a wild-type gene oran objective mutant gene as a template to prepare linear DNA of aconstruct having an introduced objective mutation. A protein wassynthesized in a cell-free synthesis reaction system derived fromEscherichia coli using this DNA as the template. A supernatant fractionafter centrifugation of a product synthesized by a dialysis method for 6hours using 1 mL of a reaction scale was purified with affinity for Nito yield an elution fraction. Subsequently, the presence of a proteinconceivable to be an objective enzyme was identified by SDS-PAGE andstaining using SYPRO ORANGE protein gel stain (Life Technologies JapanLtd.). A protein concentration in the yielded elution fraction wasquantified by the Bradford method using BSA as a standard substance. Theelution fraction was adjusted to an objective concentration as needed,and subsequently subjected to the evaluation. For the wild-type enzyme,the preparation of linear DNA, the cell-free synthesis, and thepurification and analysis of the enzyme were performed in the samemanner as above. Enzymes prepared by PCR using the wild-type gene as thetemplate are shown in Table 4. Enzymes prepared by PCR using the genecarrying the introduced objective mutation as the template are shown inTables 5 and 6. The resin and the buffer used for the purification areas follows.

Resin: Ni Sepharose High Performance (GE Healthcare Japan)

Buffer: Binding Buffer (NaCl 750 mM, NaPi 20 mM, pH8.0),

Wash Buffer (NaCl 750 mM, NaPi 20 mM, pH8.0)

Collection and measurement Buffer (NaCl 300 mM, NaPi 50 mM, EDTA 34 mM,pH7.0, 10% D₂O, 0.01% NaN₃)

Example 6 Evaluation of Activity and Substrate Specificity

The activity and the relative activity of the wild-type enzyme and themodified enzyme synthesized in Example 5 were evaluated according to themethod in Example 2. The results are shown in Tables 4, 5 and 6. Meanvalues of results from 3 samples of the wild-type were used for valuesfrom WT in Tables 4 and 5. The results in Table 6 were calculated frommean values when the experiment was performed three times for the samesample. When the modified enzyme having a plurality of introducedmutations is represented, each of the introduced mutations was markedoff using a slash and described consecutively. For example, a mutantI136R/I292F denotes a modified enzyme having two mutations of I136R andI292F. By introducing the mutation, the activity of the modified enzymewas further enhanced and/or became more equivalent for each BCAA,compared to that of WT. Compared to the case of introducing onemutation, the relative value of the activity for each BCAA became moreequivalent by introducing two mutations that made the relative valuemore equivalent than WT.

TABLE 4 Relative values of activity of modified enzymes relative to WT(left) and relative values of activity of enzymes for each BCAA(right)(1). Activity (change Relative activity of absorbance (whenactivity for Leu per one minute) is regarded as 100%) Leu Ile Val LeuIle Val I136C 258% 242% 318% I136C 100% 80% 86% I136Y 204% 218% 241%I136Y 100% 91% 83% I136A 188% 183% 221% I136A 100% 82% 82% I136G 162%157% 220% I136G 100% 82% 95% I136S 148% 150% 172% I136S 100% 86% 81%I136N 142% 137% 153% I136N 100% 82% 76% I136W 137% 152% 152% I136W 100%94% 78% I1360 108% 113% 113% I1360 100% 88% 73% I136E  97%  82%  92%I136E 100% 71% 66% I1361  95%  95%  93% I1361 100% 85% 69% I136P  92% 70%  96% I136P 100% 65% 73% I136D  55%  50%  55% I136D 100% 76% 69%I292H 245% 270% 307% I292H 100% 93% 88% I292N 241% 274% 304% I292N 100%96% 88% I292Y 234% 265% 274% I292Y 100% 96% 82% I292L 231% 218% 244%I292L 100% 80% 74% I292K 231% 248% 264% I292K 100% 91% 80% I2920 223%250% 262% I2920 100% 95% 82% I292R 211% 213% 229% I292R 100% 86% 76%I292D 208% 186% 242% I292D 100% 76% 81% I2921 202% 218% 226% I2921 100%91% 78% I292E 200% 195% 233% I292E 100% 83% 82% I292S 195% 213% 219%I292S 100% 93% 78% I292C 161% 165% 154% I292C 100% 87% 67% I292A 157%170% 175% I292A 100% 92% 78% I292G 145% 158% 171% I292G 100% 92% 82%I292V 128% 134% 133% I292V 100% 89% 73% I292W 120% 138% 117% I292W 100%97% 68% I292P  73%  63%  75% I292P 100% 73% 72% WT 100% 100% 100% WT100% 85% 70%

TABLE 5 Relative values of activity of modified enzymes relative to WT(left) and relative values of activity of enzymes for each BCAA(right)(2). Activity (change Relative activity of absorbance (whenactivity for Leu per one minute) is regarded as 100%) Leu Ile Val LeuIle Val I136M 250% 300% 370% I136M 100% 93% 102%  I136R 239% 279% 330%I136R 100% 91% 95% I136H 191% 206% 180% I136H 100% 83% 65% I136F 184%238% 277% I136F 100% 100%  102%  I136L 180% 171% 240% I136L 100% 73% 92%I136K 150% 171% 217% I136K 100% 88% 99% Il36V  75%  65%  63% Il36V 100%66% 57% I292F 268% 362% 370% I292F 100% 105%  95% WT 100% 100% 100% WT100% 76% 66%

TABLE 6 Relative values of activity of modified enzymes relative to WT(left) and relative values of activity of enzymes for each BCAA(right)(3). Activity (change of Relative activity (when activityabsorbance per one minute) for Leu is regarded as 100%) Leu Ile Val LeuIle Val 1136R/1292F 158% 214% 232% 1136R/1292F 100% 101% 94% WT 100%100% 100% WT 100%  75% 64%

A 96-well microwell plate was used in place of the cuvette having 1 cmof the optical pass length, an enzymatic reaction was performed in thefollowing reaction solution at 30° C. for one minutes, and then thechange of the absorbance at 340 nm was measured. An enzyme to be usedwas synthesized using the linear DNA prepared using the wild-type geneas the template and purified as shown in Example 5. The reactionsolution was prepared by mixing 150 μL of buffer (0.2 M HEPES, 0.28 Msodium chloride, 8.4 mM disodium hydrogen phosphate, pH 7.0 or 7.5 or8.0), 30 μL of an aqueous solution of 25 mM NAD⁺, 1.5 μL of an aqueoussolution of 1 M potassium chloride, 3 μL of an aqueous solution of 10 mML-leucine or L-isoleucine or L-valine and 100.5 μL of MilliQ water, andfurther adding 15 μL of 1 mg/mL of an enzyme solution thereto. Theresults are shown in Tables 7 and 8. Each value for I136K and I136R wasobtained from one experiment, and other values for other enzymes weremean values calculated from two experiments for the same sample. Table 7shows relative values of the activity of the modified enzymes relativeto WT, and Table 8 shows relative values of activity of the enzymes foreach BCAA.

TABLE 7 Relative values of activity of modified enzymes relative to WT.Activity (change of absorbance per one minute) pH 7.0 pH 7.5 pH 8.0 LeuIle Val Leu Ile Val Leu Ile Val I136A 258% 182% 412% 174% 164% 234% 143%176% 210% I136C 328% 233% 610% 206% 206% 314% 157% 218% 285% I136E  69% 47%  66%  40%  35%  30%  27%  25%  17% I136F 252% 270% 538% 152% 208%255% 136% 194% 214% I136G 270% 207% 488% 188% 188% 255% 153% 201% 219%I136H 292% 251% 375% 191% 223% 211% 157% 199% 186% I136K 231% 219% 436%170% 200% 236% 152% 188% 203% I136L 291% 236% 497% 185% 219% 271% 158%196% 227% I136M 317% 255% 588% 179% 240% 327% 153% 226% 277% I136N 235%199% 301% 187% 171% 176% 150% 167% 139% I136Q  84% 112% 175%  89%  96% 75%  81%  99%  68% I136R 283% 271% 579% 167% 235% 294% 145% 217% 245%I136S 166% 165% 274% 148% 151% 143% 130% 154% 123% I136T  64%  68%  77% 71%  65%  57%  69%  70%  56% I136V  76%  69%  99%  73%  66%  64%  81% 75%  68% I136W 195% 169% 221% 146% 146% 134% 119% 133% 110% I136Y 257%236% 522% 158% 193% 244% 134% 178% 201% I292A 208% 176% 309% 160% 185%180% 132% 162% 178% I292C 224% 135% 335% 177% 184% 199% 146% 168% 164%I292D 396% 339% 739% 236% 278% 359% 183% 253% 277% I292E 335% 381% 666%216% 325% 359% 162% 267% 287% I292F 396% 532% 817% 231% 397% 357% 180%327% 281% I292G 324% 296% 561% 210% 261% 296% 165% 225% 243% I292H 357%408% 756% 200% 326% 350% 158% 294% 269% I292K 372% 342% 588% 211% 296%300% 168% 249% 255% I292L 365% 380% 544% 210% 302% 282% 158% 250% 242%I292M 245% 192% 361% 180% 179% 206% 157% 183% 195% I292N 332% 463% 784%188% 375% 364% 147% 310% 294% I292Q 352% 434% 706% 207% 377% 345% 153%287% 272% I292R 380% 329% 653% 213% 316% 325% 171% 284% 257% I292S 353%363% 640% 211% 311% 326% 163% 265% 270% I292T 305% 313% 495% 200% 267%294% 153% 225% 244% I292V 136% 102% 144% 103% 105% 106% 99% 104% 106%I292W 165% 231% 194% 125% 189% 123% 116% 154% 110% I292Y 352% 481% 753%205% 358% 316% 159% 268% 231% I136F/ 143% 363% 517% 104% 218% 221%  89%158% 161% I292F I136R/ 235% 428% 591% 141% 285% 242% 115% 185% 178%I292F WT 100% 100% 100% 100% 100% 100% 100% 100% 100%

TABLE 8 Relative values of activity of enzymes for each BCAA. Relativeactivity (when activity far Leu is regarded as 100%) pH 7.0 pH 7.5 pH8.0 Leu Ile Val Leu Ile Val Leu Ile Val I136A 100%  57%  97% 100%  70%105% 100%  95% 115% I136C 100%  57% 113% 100%  75% 120% 100% 107% 141%I136E 100%  55%  58% 100%  65%  59% 100%  72%  48% I136F 100%  86% 129%100% 102% 133% 100% 111% 123% I136G 100%  52% 110% 100%  75% 107% 100%102% 112% I136H 100%  59%  78% 100%  87%  87% 100%  98%  92% I136K 100% 76% 115% 100%  88% 109% 100%  95% 104% I136L 100%  65% 104% 100%  88%116% 100%  96% 112% I136M 100%  67% 113% 100% 100% 144% 100% 114% 142%I136N 100%  68%  78% 100%  68%  74% 100%  86%  72% I136Q 100% 107% 126%100%  81%  67% 100%  95%  66% I136R 100%  77% 124% 100% 105% 139% 100%115% 132% I136S 100%  80% 100% 100%  76%  76% 100%  92%  74% I136T 100% 85%  73% 100%  69%  63% 100%  78%  63% I136V 100%  72%  78% 100%  68% 70% 100%  72%  65% I136W 100%  69%  69% 100%  74%  72% 100%  86%  73%I136Y 100%  74% 123% 100%  86% 115% 100% 103% 118% I292A 100%  68%  90%100%  86%  89% 100%  95% 105% I292C 100%  67%  91% 100%  78%  89% 100% 89%  88% I292D 100%  69% 113% 100%  88% 120% 100% 107% 118% I292E 100% 92% 121% 100% 112% 131% 100% 127% 138% I292F 100% 108% 125% 100% 128%125% 100% 141% 122% I292G 100%  73% 105% 100%  93% 111% 100% 105% 115%I292H 100%  92% 128% 100% 122% 139% 100% 144% 133% I292K 100%  74%  96%100% 105% 112% 100% 115% 119% I292L 100%  84%  90% 100% 107% 106% 100%122% 119% I292M 100%  63%  89% 100%  74%  90% 100%  90%  97% I292N 100%112% 143% 100% 149% 153% 100% 164% 157% I292Q 100%  99% 122% 100% 136%132% 100% 145% 139% I292R 100%  70% 104% 100% 110% 120% 100% 129% 118%I292S 100%  83% 110% 100% 110% 122% 100% 126% 130% I292T 100%  82%  98%100% 100% 116% 100% 113% 124% I292V 100%  60%  64% 100%  76%  81% 100% 81%  84% I292W 100% 113%  71% 100% 112%  77% 100% 103%  74% I292Y 100%110% 130% 100% 131% 122% 100% 131% 114% I136F/ 100% 203% 219% 100% 155%168% 100% 137% 141% I292F I136R/ 100% 147% 153% 100% 151% 136% 100% 125%122% I292F WT 100%  80%  61% 100%  75%  79% 100%  77%  78%

Example 7 Evaluation of Thermal Stability

Five solutions of LeuDH WT and the following modified enzymessynthesized in Example 5 were treated with heat at 60, 70 and 80° C. forone hour, and subsequently the activity was measured. A remainingactivity of each enzyme solution after the treatment with heat is shownin Table 9. The remaining activity of any modified enzyme after thetreatment with heat at 60° C. or 70° C. was higher than that of WT.

TABLE 9 Relative activity after treatment with heat at indicatedreaction temperature when activity before treatment is regarded as 100.WT I292F I136K I136F I136R I136M 60° C. 74 92 86 90 91 96 700° C.  58 6267 74 75 74 80° C. 0 0 0 0 0 1

INDUSTRIAL APPLICABILITY

The modified enzyme of the present invention is useful for the rapidmeasurement of the total branched-chain amino acid concentration. Themodified enzyme of the present invention is also useful for themeasurement of any branched-chain amino acid and/or the production ofderivatives of any branched-chain amino acid. The modified enzyme of thepresent invention is further useful as a liquid reagent. The analysismethod of the present invention is useful for the diagnosis of diseasessuch as cirrhosis or hepatic encephalopathy.

While the invention has been described in detail with reference topreferred embodiments thereof, it will be apparent to one skilled in theart that various changes can be made, and equivalents employed, withoutdeparting from the scope of the invention. Each of the aforementioneddocuments is incorporated by reference herein in its entirety.

1. A modified leucine dehydrogenase enzyme comprising at least one aminoacid mutation as compared to a non-modified leucine dehydrogenaseenzyme, wherein said modified leucine dehydrogenase is improved in oneor more properties selected from the group consisting of: (a) substratespecificities for total branched-chain amino acids; (b) activity for anybranched-chain amino acid; (c) thermal stability, and (d) combinationsthereof.
 2. The modified leucine dehydrogenase enzyme according to claim1, wherein the mutation is a substitution of isoleucine in a TGI motifin an amino acid sequence of the non-modified leucine dehydrogenaseenzyme.
 3. The modified leucine dehydrogenase enzyme according to claim2, wherein the isoleucine in the TGI motif is substituted with an aminoacid selected from the group consisting of methionine, arginine,histidine, phenylalanine, leucine, lysine, cysteine, tyrosine, alanine,glycine, serine, asparagine, and tryptophan.
 4. The modified leucinedehydrogenase enzyme according to claim 1, wherein the mutation is asubstitution of isoleucine in a GVI motif in an amino acid sequence ofthe non-modified leucine dehydrogenase enzyme.
 5. The modified leucinedehydrogenase enzyme according to claim 4, wherein the isoleucine in theGVI motif is substituted with an amino acid selected from the groupconsisting of phenylalanine, histidine, asparagine, tyrosine, leucine,lysine, glutamine, arginine, aspartic acid, threonine, glutamic acid,serine, cysteine, alanine, glycine, valine, tryptophan, and methionine.6. The enzyme comprising at least one amino acid mutation according toclaim 1, wherein the non-modified leucine dehydrogenase enzyme isderived from Geobacillus stearothermophilus.
 7. The modified leucinedehydrogenase enzyme according to claim 1, comprising a protein selectedfrom the group consisting of: (A) a protein comprising the amino acidsequence of SEQ ID NO: 2, but having a substitution of isoleucine in theTGI motif with an amino acid selected from the group consisting ofmethionine, arginine, histidine, phenylalanine, leucine, lysine,cysteine, tyrosine, alanine, glycine, serine, asparagine, andtryptophan, (B) a protein comprising the amino acid sequence of SEQ IDNO: 2, but having a substitution of isoleucine in the GVI motif with anamino acid selected from the group consisting of phenylalanine,histidine, asparagine, tyrosine, leucine, lysine, glutamine, arginine,aspartic acid, threonine, glutamic acid, serine, cysteine, alanine,glycine, valine, tryptophan, and methionine, (C) a protein comprisingthe amino acid sequence of SEQ ID NO: 2, but having a substitution ofisoleucine in the TGI motif with an amino acid selected from the groupconsisting of methionine, arginine, histidine, phenylalanine, leucine,lysine, cysteine, tyrosine, alanine, glycine, serine, asparagine, andtryptophan, and a substitution of isoleucine in the GVI motif with anamino acid selected from the group consisting of phenylalanine,histidine, asparagine, tyrosine, leucine, lysine, glutamine, arginine,aspartic acid, threonine, glutamic acid, serine, cysteine, alanine,glycine, valine, tryptophan, and methionine, and (D) a protein asdescribed in (A), (B), or (C) above, but also comprising one or severaladditional mutations of amino acid residues, and having one or moreimproved properties selected from the group consisting of: (a) substratespecificities for total branched-chain amino acids; (b) activity for anybranched-chain amino acid; and (c) thermal stability.
 8. A method ofanalyzing total branched-chain amino acids, comprising measuring thetotal branched-chain amino acids contained in a test sample using themodified leucine dehydrogenase enzyme according to claim
 1. 9. Themethod according to claim 8, comprising mixing the test sample withnicotinamide adenine dinucleotide (NAD⁺) and detecting NADH formed fromNAD⁺ by an action of the modified leucine dehydrogenase enzyme.
 10. Amethod of producing a derivative of a branched-chain amino acid,comprising forming the derivative from the branched-chain amino acidusing the modified leucine dehydrogenase enzyme according to claim 1.11. A polynucleotide encoding the modified enzyme according to claim 1.12. An expression vector comprising the polynucleotide according toclaim
 11. 13. A transformant comprising the expression vector accordingto claim
 12. 14. A method of producing a modified enzyme, comprisingforming the modified enzyme using the transformant according to claim13.
 15. A kit for analyzing total branched-chain amino acids, comprisingthe modified enzyme according to claim
 1. 16. The kit for analyzing thetotal branched-chain amino acids according to claim 15, furthercomprising at least one of a buffer solution or a buffer salt for areaction and nicotinamide adenine dinucleotide (NAD⁺).
 17. An enzymesensor for analyzing total branched-chain amino acids, comprising (a) anelectrode for detection and (b) the modified enzyme according to claim1, which is immobilized or retained on the electrode for detection.